Radioimmunoassay for measurement of thyroxine (T{HD 4{B ) and triiodothyonine (T{HD 3{B ) in blood serum

ABSTRACT

This invention relates to a highly accurate, rapid and simple estimation of thyroxine (T4) directly from blood serum and also relates to the accurate measurement of triiodo-L-thyronine (T3) directly from blood serum. More specifically, the invention relates to a rapid, specific and reliable radioimmunoassay (RIA) technique for measurement of both T4 and T3 in unextracted serum. The method requires very small amounts of serum, e.g., 25 microliters ( Mu l) to measure T4 concentration in nearly all specimens representing clinical states of eu-, hypo- and hyperthyroidism, and 250 Mu l to measure T3 concentrations in specimens representing most clinical states.

United States Patent 1 Chopra RADIOIMMUNOASSAY FOR MEASUREMENT OF THYROXINE (T AND TRIIODOTHYONINE (T IN BLOOD SERUM [75] Inventor:

[73] Assignee: Professional Staff Association of the Los Angeles County Harbor General Hospital, Torrance. Calif.

[22] Filed: June 23, 1972 [21] Appl. No: 265,586

lnder J. Chopra, Torrance, Calif.

1 1 Oct. 7, 1975 3,714,344 1/1973 Brown 424/1 1745,21] 7/1973 Brown et al. 424/1 3,749,556 7/1973 Barak et a1. .1 23/252 Primary ExaminerLeland A. Sebastian Attorney, Agent or Firm-l. Morley Drucker ABSTRACT This invention relates to a highly accurate, rapid and simple estimation of thyroxine (T directly from blood serum and also relates to the accurate measurement of triiodo-L-thyronine (T directly from blood serum. More specifically, the invention relates to a rapid specific and reliable radioimmunoassay (RlA) technique for measurement of both T and T in unextracted serum. The method requires very small amounts of serum, 6g, 25 microliters .1.1) to measure T concentration in nearly all specimens representing clinical states of eu-, hypoand hyperthyroidism, and 250 1.1 to measure T concentrations in specimens representing most clinical states,

11 Claims, 3 Drawing Figures RADIOIMMUNOASSAY FOR MEASUREMENT OF THYROXINE (T AND TRIIODOTHYONINE (T IN BLOOD SERUM The invention described herein was made in the course of work under a grant or award from the Department of Health, Education and Welfare.

BACKGROUND OF THE INVENTION In a normal human serum, about 99.97% ofthyroxine (T and 99.7% of triiodothyronine (T present, is bound to proteins such as thyroxine-binding globulin (TBG), prealbumin (TBPA) and albumin; the remain ing 0.03% of T and 0.371 of T is present as unbound (dialyzable or free) hormones. The serum concentration of thyroid hormones, among other facts such as thyroidal secretion and degradation, is also dependent on serum concentration of THC; other binding proteins in serum are relatively less important since their binding affinity for thyroid hormones is far less than that of TBG. The concentration of TBG is elevated in conditions such as pregnancy, estrogen treatment or a genetic abnormality; serum total T (and T is increased in these situations. Conversely, serum TBG concentration is decreased during treatment with androgens or due to a genetic deficiency of TBG. This is associated with subnormal concentrations of thyroid hormones. However, in either of the above-mentioned situations of altered TBG concentration. the patient remains eumetabolic and concentrations of free T, (and T and that of total T (and T corrected for TBG abnormality are within the range of normal subjects.

Until a few years ago, serum T concentration was assessed indirectly by measurements of organic (protein bound or butanol extractable) iodine. However, these measurements were frequently erroneous because of iodide contamination in the laboratory or of administration of iodide containing drugs to the patients. In order to overcome these problems, a competitiveprotein binding assay (CPBA) for T was introduced by Murphy and Pattee (I). This procedure employs the principal of saturation analysis, and quantitates T relatively specifically. by measurement of displacement of radioactive T, from T -binding sites on TBG. It allows measurements of T in a range of 3-20 ug7( using a hut-anol-ethanol extract of serum. In the Murphy- Pattee method, one must first extract thyroxine from the blood serum with alcohol or butanol-ethanol; this introduces error since the extraction procedure itself results in the extraction of varying amounts of thyroxine. The drying of the extract is also time consuming, and must be accomplished before one commences the measurement of T,,. (l) Murph} and Pattec J. Lab. Clin. Med. 66.16] July I965 The Murphy-Pattee procedure thus leaves one with a dried extract containing T; as well as much nonthyroxins, such as lipids. To the tubes containing the dried extract, one than adds a l/32 diluted human serum containing radioactive T (T The T in the dried extract competes with T for binding sites on TBG. After equilibrium is reached one separates the unbound T by means of an ion exchange resin and measures the amount of radioactive counts (of T remaining bound to TBG. The quantitation of T is accomplished by reading from a standard curve, prepared simultaneously with known amounts of T The disadvantages of the Murphy-Pattee method are that it requires an extraction procedure; it is not sensitive enough and it is cumbersome for processing a large number of samples.

A radioimmunoassay (RIA) has been previously developed and reported, by the applicant herein in con junction with others, which employs a highly specific antibody to T for its subsequent measurement instead of utilizing TBG from human serum. hi this method, a rabbit anti-thyroglobulin antiserum is employed as the T binding protein. Further, in this method, the T,,must be first extracted as in the Murphy-Pattee method but the method is more sensitive than Murphy-Pattee. This RIA method of applicant herein is reported, in detail, in Journal of Clinical Endrocrinolugy, 33:865.]971, in corporated herein by this reference. In general, the precision, reproducibility and practicality of this RIA is comparable to those of competitive protein binding assay (CPBA) using serum TBG as the T binding protein.

SUMMARY OF THE INVENTION l have found that I can achieve significantly improved measurement of T and T directly from unextracted serum by displacing T and T from serum thyroxine (or T binding globulin (hereinafter generically referred to as TBG by means ofa foreign compound (i.e., one not itself a thyroid hormone) which compound not only has the capability of preventing completely the binding of radioactive T and radioactive T (T or T,,*, respectively) or T and T to TBG, respectively, but does not inhibit in any way the reaction of T, and T with T antibodies and T -antibodies used as the hormone binding proteins (instead of TBG). Foreign compounds falling in this category will be termed herein, and in the claims simply as blocking agent" or blocking agents.

The novel approach adopted herein is believed to be broadly new and is not dependent upon the use of a particular blocking agent. Many different types of blocking agents have been proven to have the desired qualities aforementioned, e.g., 8-anilino-l-napthalenesulfonic acid (ANS), 3-(4 anilino-l-napthylazo) 2,7- napthalene disulfonic acid (ANNDS), 2,4 fi-trinitro benzene sulfonic acid (TNBS), napthalene sulfonic acid, Thimerosal, 5-5-diphcnyl 2thiohydantain, Doxepin HCl, Diazepam, sodium salicylate, prochlorperazine, halofenate (MK- l Merck, Sharpe and Dohme.

Further, the inhibition of the binding of T (or T to thyroxine binding prealbumin (TBPA) is accomplished by the setting up of the assay in a suitably buffered me dium, e.g., containing barbital ions, e.g., barbital buffer, and having a pH of between about 6.8-9.6. T binding by prealbumin is also affected by the presence of compounds such ANS, or sodium salicylate.

Thus, with respect to the measurement of T by my novel RIA, the process involves only the following few steps: l incubation for l hour, of human serum, in the presence of radioactive T T antibody, and one of the blocking agents mentioned above or others in a barbital or other suitable buffer to thereby displace T bound to TBG in the serum and make it available for reaction with T antibody (and thus measurable by RIA techniques) while minimizing or completely inhibiting the binding of the added radioactive T to TBG. Finally, the radioactive T bound to the antibody is separated from free radioactive T This may be accomplished in various ways, e.g., by use of a second antibody", e.g., goat antirabbit gammaglobulin which precipitates the antibody bound radioactivity which may then be counted. A standard curve is prepared with known amounts of T and unknowns are read off the curve. Using the just-described RIA, it is feasible to quantitate T in a wide range (I to 40 ug7r) using only l to ul of unextracted serum.

Measurement of T in serum has been more involved and difficult than that of T,,. On an average, T is present in normal serum in concentrations about 1/70 that of T However, the importance of its quantitation in serum has been highlighted by recent suggestions that T may be the predominant biologically active thyroid hormone and that T exerts its biological effect only after prior conversion to T in the body. Several instances of hyperthyroidism due to elevated serum T;, (and normal serum T have been described. A popular method for measurement of T involves three steps. i.e., extraction of thyronines from serum by column chromatography. separation of T, from T by paper chromatography and finally quantitation of T;, by competitive protein binding assay using TBG in a manner similar to that for T measurement by Murphy-Pattee. However. chromatographic separation of T from T may not always be complete. It may also be associated with in vitro conversion of T to T thereby leading to inappropriate high values. These problems have been circumvented by the use ofa radioimmunoassay (RIA) method for quantitating T in unextracted serum by a method similar to that abovedescribed for T assay.

Thus. improvements in accurate measurement of serum T;, by RIA requires, according to this invention. adequate blocking of thyroninebinding globulin (TBG) in serum, by means of the same blocking agents heretofore enumerated. The approach to T measurement is precisely the same as for T measurement; the improvement in the T RIA being due primarily to the use of blocking compounds such as ANS, which block '1}, binding sites. ANS. when used in a concentration of l ug/ul. test serum. displaces nearly all T;, bound to T86 and prevents completely the binding of radioactivc T, to TBG. ANS has negligible affinity for T -binding sites on the rabbit antiserum used in this RIA (which antiserum was produced by immunization with T -enriched thyroglobulin). The T -antibody may be precipated with a second antibody and the bound radioactive T is separated from tree T;,. and counted by previously known methods, to provide an accurate measurement of T BRIEF DESCRIPTION OF THE DRAWINGS FIG. I is an example of the standard curve relating T, (ng) to percent radioactive T precipitated;

FIG. 2 illustrates two more standard curves indicating the substantially completely blocking ofTBG by the particular blocking agent utilized, viz. ANS; and

FIG. 3 is an example of the standard curve relating to T (rig) to percent radioactive T, precipitated DESCRIPTION OF THE PREFERRED EMBODIMENTS A. T Assay In general, the method requires the incubation for about one hour of human serum, in the presence of a barbital or other suitable buffer and in the presence of T, antibody. TR. and a blocking agent present in sufficient quantity to displace T bound to TBG in the serum and make it available for reaction with the said T antibody. One can readily assess the amount of any particular blocking agent required to meet the foregoing condition. Thus, by way of example only, the ability of ANS to displace T, has been demonstrated by its displacement of radioactive T from TBG in a CPBA system, (1 supra. ug of ANS was found to displace l8 nanograms (rig) T Thus, when 100 ug of ANS is added to 25 ul of serum. it will displace all T up to a concentration of 72 ug per 100 ml. of serum. Even though the upper concentrations of T found are well below 72 ug/IOO ml, an excess of ANS, e.g., ug added to 25 ul of serum, may be and is used, in the instant assay procedure. In general, then, it is well within the skill of the art, to determine the required minimal quantity of blocking agent required for the assay procedure. Further examples of the displacement ability of other blocking agents are set forth in Table I below, the first compound set forth in Table I being ANS for ready comparison.

TABLE I AMOUNT T DISPLACED BLOCKING AGENT FROM TBG (ng) lUtl ug utilized except where noted otherwise) I. ANS

ANNDS (51) ug] TNBS uNapthalenc Sulfonic Acid 5. 5 diphenyl2 thiohydantoin Doxepin HCl Diazepam Prochlorpcrazine Dilantin 1 himcrosal l mg.) Sodium Salicylatc ll) mg.) Halofcnate* tMK-l85J (hlorpromayine HCI (5U ugl Proprietary compound supplied by Merck, Sharp 81 Dohnic The amount of Ti added is determined by that quantity required to give to the assay tube, in which the assay is being conducted, a measurable counting rate, after reaction with the T antibody. The amount of radioactive T to be added is not critical and may vary over a wide range depending upon the sensitivity of the counting equipment. One may, for example, utilize sufficient radioactive T initially, to cause a counting rate of 2()()O-6()()() CPM (counts per minute) to result from measurement of the Tfl-antibody precipitate. One may also desire to measure the free radioactive T in which event, the counting rate of the free radioactive T, should be preferably at this range. Of course, as the counting equipment becomes more sensitive, the amount of radioactive T to be added may be further reduced.

Preparation of antibodies specific to T is known and will be described in some detail hereafter. The amount of T -antibody added, to a predetermined quantity of human serum, e.g., 25 ul is that quantity having the ability to bind substantial quantities of T e.g.. from about 20-60% of radioactive T in the absence of any non-radioactive T The contents of the assay tubes are buffered, preferably by barbital ions. to a pH in the range of from about 6.8 to 9.6. with a pH of about 8.6 being preferred.

Incubation of the contents of the assay tubes. containing measured amounts of T.,*. T, antibody, and blocking agent in a measured amount of serum, takes place over a period of an hour or so at room tempera ture and for minutes at 4 C. During this period of incubation, competitive reactions between TR and T on the one hand, with T, antibody on the other, takes place, and near equilibrium of these competing reaction is attained.

The radioactive T bound to antibody (antibody bound radioactivity) is then separated from the free radioactive T, by anyone of a number of methods. For example, a charcoal suspension when added to the final reaction mixture, after the end of the incubation period, adsorbs the free radioactivity. The reaction mixture, containing the charcoal, is centrifuged and the charcoal settles. One may count either the bound radioactivity in the supernatant, or the free radioactivity in the charcoal.

Alternatively, one may add a polyethylene glycol (Carbowax 6000) to the reaction mixture. Polyethylene glycol will precipitate gammaglobulins, and when added to the reaction mixture, will precipitate the antibody bound radioactivity, and may be measured as indicated above.

The method of precipitation of antibody bound radioactivity presently preferred, involves the use of a second antibody which will precipitate the antibody bound radioactivity. After a suitable period of incubation. e.g., 24 hours at 4 C., the antibody bound radioactivity may then be counted. Measurement of T concentration is made by correlating the antibody found radioactivity with a standard curve previously prepared.

B. T;, Assay In general. the method of measuring T requires the incubation for about hours of human serum, in the presence of a barbital or other suitable buffer and in the presence of T antibody, T;,*, and a blocking agent, present in sufficient quantity to displace all T bound to TBG in the serum and make it available for reaction with said T, antibody. It has been demonstrated that TBG competes with T antibody for radioactive T;, as well as for non-radioactive (or stable) T The concentration of blocking agent required to prevent this competition or interference from TBG is readily determined. ln general, the binding of T to TBG is much weaker than is T, to TBG and the amount of blocking agent required is less per given amount of human serum than is required for the T assay. Thus, for example, l ug/ul serum of ANS is utilized in a T assay as compared with 4-6 ug/ul ANS for a T assay. The ability of ANS to displace T has been demonstrated by its displacement of radioactive T from TBG. l0 ug. of ANS was found to displace l 1.2 nanograms (ng) T Thus, when 200 ug. of ANS is added to 250 ul of serum, it may displace all T up to a concentration of 88 ug T per 100 ml. of serum. Even though the upper concentrations of T found are well below 2.5 ug/lOO ml. an excess of ANS. e.g., 250 ug is normally added to 250 ul of serum in the instant assay procedure. ln general, then, it is well within the skill of the art, to determine the required minimal quantity of blocking agent required for the assay procedure. The procedure for determining the minimal quantity for ANS is set forth in Example 2 hereof. Further, examples of the displacement ability of other blocking agents are set forth in Table ll below, the first compound set forth in Table II being ANS for ready comparison.

TABLE II AMOUNT T DISPLACED BLOCKING AGENT FROM TBG (ng) 10 ug utilized except where noted otherwise) I. ANS l 1.2

3. ANNDS [0.4

3. TNBS ltXl ug.) 7.0

4. aNapthalene Sulfonic acid 7.0

i 100 ug) 5. 5,5 diphenyl-2 thiohydantoin 7.6

6. Doxepin HCI lttX 7. Diazepam 7.0

8. Prochlorperazine l .t)

9. Thimerosal I00 ug.) 8.0

it). Sodium Salieylate mg) l Ll) l l. Halofenatc* (MK-I) 5.2

12. Chlorpromazine HCI (25 ug) 6.6

*Proprictar compound supplied by Merck, Sharp A; Duhme.

The amount of T added is determined by that quantity required to give to the assay tube, in which the assay is being conducted, a measurable counting rate, after reaction with the T antibody. The amount of radioactive T to be added is not critical and may vary over a wide range depending upon the sensitivity of the counting equipment. One may, for example, utilize sufficient radioactive T initially, to cause a counting rate of 2000600() CPM (counts per minute) to result from measurement of the T -,*-antibody precipitate. One may also measure the free radioactive T in which event, the counting rate of the free radioactive T should be preferably at this range. Of course. as the counting equipment becomes more sensitive, the amount of radioactive T to be added may be further reduced.

Preparation of T antibodies specific to T is known and will be described in some detail hereafter. The amount of T;, antibody added, to a predetermined quantity of human serum, e.g., 250 ul, is that quantity having the ability to bind from about 2060"/( of radioactive T;, added in the absence of any non-radioactive T The contents of the assay tubes are buffered, preferably by Barbital ions, to a pH in the range of from about 6.8 to 9.6, with a pH of about 86 being preferred.

Incubation of the contents of the assay tubes, containing measured amounts of T T antibody, and blocking agent in a measured amount of serum, takes place over a period of 20 hours or so, at 4 C. During this period of incubation, competitive reactions between T and T on the one hand, with T antibody on the other, takes place, and near equilibrium of these competing reactions is attained.

The radioactive T bound to antibody (antibody bound radioactivity) is then separated from the free ra dioactive T; by anyone ofa number of methods. For ex ample, a charcoal suspension when added to the final reaction mixtures, after the end of the incubation period, adsorbs the free radioactivity. The reaction mixture. containing the charcoal, is centrifuged and the charcoal settles. One may count either the bound radioactivity in the supernatant, or the free radioactivity in the charcoal.

Alternatively, one may add a polyethylene glycol (Carbowax 6000) to the reaction mixture. Polyethylene glycol will precipitate gammaglobulins, and when added to the reaction mixture, will precipitate the antibody bound radioactivity, and may be measured as indicated above.

The method of precipitation of antibody bound radioactivity presently preferred. involves the use of a sec ond antibody which will precipitate the antibody bound radioactivity. The antibody bound radioactivity is then counted and measurement of T concentration is made by correlating the antibody bound radioactivity with a standard curve previously prepared.

C. Specific Examples EXAMPLE 1 The following example illustrates the use of ANS as a blocking agent in the measurement of T. at a variety of concentrations, and illustrates. as well, the making of a standard curve. to allow accurate correlation of the unknown T concentrations with the standard curve. This example serves as the basis for a paper to be shortly published in Journal of Clinical Endoclzrinology. 34: 938 1972 under the title A Radioimmunoassay For Measurement of Thyroxine in Unextracted Serum".

The reagents employed are:

1. T -binding antiserum or T, antibody: The serum used was obtained from a rabbit immunized with normal human thyroglobulin (Tg), as described by Chopra et al.. J. Clinical limlm'rinulogv 32:299. It was used in a final dilution of 1:300; in this dilution it bound approximately 509 of a tracer amount of radioactive T Radioactive 1) T (SA 5075 uc/ug) in 50% propylene glycol was obtained from Abbott Laboratories. North Chicago. Ill.

3. ANS was obtained from K 64 K Laboratories. Hollywood. Calif.

4. Reagent grade Na L-T (non radioactive T was obtained from Mann Research Laboratories, New York. It was weighed and dissolved in 0.1M NaOH and diluted to desired concentrations in .075M barbital buffer. pH 8.6. containing 29? normal rabbit serum (NRS The NRS is employed as a carrier protein to render the antibody precipitate visible. in the final step. but is not present in sufficient quantity to interfere with the T measurement.

The steps of the radioimmunoassay (RlA) procedure follows:

In l 75 mm disposable glass tubes. the various reagents were added in the following order to yield a final volume of 0.5 ml:

1. Three hundred ul of .075 M barbital buffer which contained 2"? NRS, I50 ug ANS and approximately 10.000 CPM (counts per minute) of "'l-T, (-02 ng T The stock solution of ANS (50 mg per I00 ml of buffer) was made fresh before every assay.

One hundred ul of various dilutions of T i.e. 0.5 ng/ml to 200 ng/ml. were employed to yield 0.05 to ng T, for a 10 to l4 point standard curve (see FIG. I].

In the case of the unknown. ul of serum was employed. followed by 75 ul of 0.075 barbital buffer containing 2'22 NRS but without additional ANS or l-T The standard curve and unknowns were assayed at least in duplicate.

3. To all tubes. 100 ul of a 1:60 dilution of T. antibody was added.

Steps 1 and 3 were conveniently and accurately performed using an automatic pipettor (Repipet 1.0 ml, Lab Industries, Berkeley. Calif. All tubes were briefly swirled after steps 2 and 3. The tubes were then incubated for 1 hr at room temperature and 5 min at 4C. Pilot experiments had indicated that a state of nearequilibrium was reached during this period of incubation.

4. To precipitate l-T., bound to antibody. approximately 40-50 ul of a previously titered goat antirabbit y globulin was added and tubes were reincubated overnight 20 hr) at 4C. The details of separation of bound from free radioactivity, correction for nonspecific binding or trapping of l- T, in the precipitate and plotting of standard curves have been described recently in a RlA for triiodothyronine (T:i)- Chopra et al. Radioimmunoassay for measurement of triodothyronine in human serum, J. Clinic Invesrg. 50:2033, October 1971.

The results are set forth below:

FIG. 1 shows the typical standard curve obtained. The curve is essentially linear between 0.3 to 10 ng, allowing measurement of serum T over the range of 1 .2 to 40 ug per 100 ml when 25 ul serum was assayed. The index of precision (A) was 0.063 in this and another standard curve.

FIG. 2 shows a comparison of standard curves obtained in barbital buffer and in the presence of 25 ul of T -free serum. The two standard curves were nearly superimposable, indicating that TBG in the serum was adequately blocked under the assay conditions used.

Table III illustrates the results of serum T as determined by the RlA described in this example and those obtained by CPBA in sera from euthyroid subjects and patients with or without thyroid functional abnormali tIes.

Mean 1 SFM ln euthyroid individuals the mean serum T; by this RlA was [0.9% higher than that by CPBA. This difference was statistically significant. It is attributable, in part, to losses in T, during extraction of serum in CPBA, since the recovery of radioactive T in the butanol-ethanol extraction averaged 88%.

Precision of T measurements by the RlA, described herein, was assessed by comparing the duplicates within assays. The mean value for percent departure of duplicates from their mean in sera was 4.32 i 0.38.

Reproducibility of estimates of serum T by RIA was studied by comparison of T concentration in 10 sera measured in duplicate in different The mean value for percent departure of duplicates from their mean was 7.1 i l .22. The working time involved in setting up an assay comprised of 94 tubes was only 1 16 min. This includes 56 min. spent in pipetting standards and test sera which would be common to all methods.

The RlA proposed here is adequately sensitive. pre' cise and reproducible. The requirement of only 50 ul of sample for duplicate determinations of serum T over a range of [.2 to 40 ug per ml, in one attempt.

not only makes the assay useful for routine clinical purposes but also for measurement of T, in serum of infants and small experimental animals where sample availability may be limited. Since T -binding antisera (a T, antibody) can be raised quite regularly when rabbits are immunized with T3 in Freunds adjuvant, this RlA appears to he a very practical method for measurement of serum T The practicality of RlA is further emphasized by the simplicity of the procedure described he re as well as the short working time involved.

The specificity of the T -binding antiserum is also quite acceptable.

EXAMPLES 2-13 The procedure of Example I may be followed, except that the following compounds listed below may be employed instead of ANS. The amounts used will be proportional to the blocking power of the compounds as listed in Table I, taking into account solubility considerations. Thus, 2 ug ANNDS per ul of serum can be successfully employed. The results should be comparable to those of Example I. 8n

EXAMPLE COMPOUND ANNDS TNBS aNaphthalene Sulfonic Acid 5,5 diphenyl-Z thiohydantoin Doxcpin HCI Diazepam Prochlorpcrazine Diluntin Thimerosal Sodium Salicylate Halofenutc* (MKJSS) (hlorpromazine HCI Proprietary compound supplied by Merck, Sharp 8; Duhnic.

EXAMPLE 14 The following example illustrates the use of ANS as a blocking agent in the measurement of T at a variety of concentrations, and illustrates, as well, the making ofa standard curve, to allow accurate correlation of the unknown T concentrations with the standard curve. This example serves as the basis for a paper to be shortly published in the Journal of Laboratory and Clim' cu! lnresligarion under the title: An Improved Radioimmunoassuy of Triiodothyronine in Serum.

The reagents employed are:

1. T -thyroglobulin conjugate was first prepared in order to make T antibody. The procedure was as follows: L-triiodothyronine (T was conjugated to human thyroglobulin (Tg) by a modification of the method of Oliver et al.,: The measurement of digitoxin in human serum by radioimmunoassay, J. Clin. Invest. 47:1035-1042, 1968. The method of preparation of human Tg was the same as described earlier. Chopra et al.: Production of anti bodies specifically binding triiodothyronine and thyroxine, J. Clin. Endoc'r. 32:299-308, 1970. To 100 mg. of Tg in 2 ml. of phosphate buffered saline (0.14 M sodium chloride, 0.01 M sodium phosphate, pH 7.5, PBS), was added mg. of Na-l-T (Mann Research Laboratories. New York) dissolved in 2 ml. of dimethyl formamide and mg. of 1-cyclo-hexyl-3 (2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (Morpho-CDI,

Aldrich Chemical Co.. Inc., Milwaukee. Wis. The

solution was kept at room temperature in the dark with occasional stirring for 18 hours. The reaction mixture was then dialyzed against 3 changes of normal saline, each time using 4 l for 24 hours at 4 C. The conjugate was stored frozen (-1(]" C). Pilot experiments using radioactive T indicated that 60% of the T was incorporated into the conjugate. 2. T -binding antiserum was then prepared. The

serum used in RIA was obtained from a New Zealand rabbit immunized with 8 injections of the aforedescribed Tg-T conjugate, 1 to 2 mg each,

emulsified in 1 ml. of complete Freund's adjuvant,

at 2-3 week intervals. One hundred ul of a 1:400

dilution of antiserum was used in a final reaction mixture of 1 ml. At this final dilution of 1:4000 it bound 33% of a tracer amount (0.1 ng) of radioactive T3.

3. In order to make the standard curve (FIG. 3) hypo-thyroid sheep serum was employed. This sheep serum was obtained from an adult sheep 6 weeks after a surgical total thyroideetomy. Total T in this sheep serum, as measured by a sensitive RlA for T was less than 1.0 ug per 100 ml. T -binding capacity of the thyroxine-binding globulin (TBG) of sheep serum was [7.5 ug per 100 ml.

4. Radioiodinated 1) T (SA 100 uci/ug was obtained from Industrial Nuclear Co., Inc., St. Louis, Mo.

5. Reagent grade Na-l-T (non-radioactive) was obtained from Mann Research Labs, New York. It

was dissolved and diluted to a concentration of ug/ml in 0.01M NaOH containing 20)? propylene glycol. Dilutions for use in standard curve, i.e., 0.1

ng/ml to 100 ng/ml T were made in 0075M barbi tal buffer, pH 8.6, containing a 171 normal rabbit serum and 0.17: sodium azide. Hereafter, this diluent is referred to as barbital buffer.

. ANS was obtained from K&K Laboratories, Hollywood, Calif. The steps of the radioimmunoassay procedure follow In l0 75 mm disposable glass culture tubes, the various reagents were added in the following order: (a) Barbital buffer, volume to adjust to a final volume of 1 ml; (b) ANS, 250 ug (100 ul of a solution containing 2.5 mg/ml); (c)

250 ul of hypothyroid sheep serum in the standards and an equal volume of test serum in all other tubes: (d) various volumes of four dilutions, i.e.,

0.1 ng/ml, 1.0 ng/ml. 10.0 ng/ml and 100 ng/ml, of

non-radioactive T to provide 10 pg to 10 ng T in tubes for a standard curve (FIG. 3); (e) 100 ul of 1:400 dilution of T -binding rabbit serum; (f) approximately 7000 cpm of T l (-01 to 0.15 ng T in 100 ul of barbital buffer.

After a brief mixing, the tubes were incubated at 4 C for 24 hours. To precipitate T l bound to rabbit anti-T 75 ul of a previously titered goat anti-rabbit y -globulin was added, and the tubes reincubated at 4 C for 20-24 hour. The details of subsequent separation of bound from free radioactivity, correction for nonspeci' fic binding or trapping of T;, 1 in the precipitate and plotting of the standard curve have been described pre viously. Chopra et al.: Radioimmunoassay for measurement of triiodothyronine in human serum, J. Clin. Invesl. 50:2033-2041, 1971.

The results are set forth below:

FIG. 3 shows a typical standard curve obtained in the presence of 250 ul sheep serum and 250 ug ANS. The threshold was 50 pg in this assay and varied between 30 and 50 pg in other assays. corresponding to a T concentration of l2 and 2t) ng/llltl ml, respectively.

Adequacy of the amount of ANS employed in RIA It has been previously demonstrated that T;, -binding proteins in serum. such as TBG. interfere in RlA of T;; by competing with T -binding antibody for radioactive (and stable) T The concentration of ANS required to prevent this interference was determined by adding to 500 ug ANS to tubes containing the typical reaction mixture but no stable T3; the proportion of T; l bound to T -binding antibody was compared to that bound to antibody in the absence of sheep serum and ANS. Radioactivity bound to antibody in the presence of 200 ug of ANS was only slightly less (95-98%) than that bound to antibody in plain buffer, indicating thereby an almost complete neutralization of the inhibiting effect of sheep serum TBG. An excess of ANS, ie, 250 ug. was employed in the final RIA procedure.

Serum T concentration in health and disease Table IV presents the data on serum T concentration in 148 subjects of whom 96 were healthy and euthyroid. 3t) hyperthyroid. l2 hypothyroid and 1t) euthyroid with elevated serum TBG either due to estrogen treatment or to a genetic abnormality.

TABLE IV Scrum T; concentration in health and disease.

In the euthyroid subjects serum T varied from 45 to 216 ng per lUU ml with mean i S.E.M. of l 12.81529 ng per lUt) ml. The serum T concentration of hyperthyroid patients was 490.7:423 ng/lOO ml. In 12 hypothyroid subjects serum T; ranged from l2 to I04 ng/ 00 ml (mean. 40. l:7.65 serum TSH as measured by an RIA in eight of these patients ranged between 62.5 and 280 uu/ml (normal range 1 to l() uu/ml). In It) sera from euthyroid subjects with high serum TBG, T was l57.6:3 l .2 ng/lOt) ml; serum T in these sera. measured by the method of Murphy et al: (The determination of thyroxine by competitive protein binding analysis employing an anion exchange resin and radiothyroxine, J. Lab. Clin. Merl. 66:161-167, 1965) was l4.8:l.42 ug per 100 ml, range lU22.5 (normal 4l l The maximal T -binding capacity of TBG as measured in serum of 7 of these subjects by the method of lnada and Sterling (lnada, M. and Sterling, K.: Thyroxine transport in thyrotoxicosis and hypothyroidism, J. (/in. Invest. 46:l422l45(), V967,) ranged between 40.7 and 56.3 ug per 100 ml (mean 46.9).

2 Gross. l, PittRivcrs. R. and Trotter. W.R.: Effect of 3:5:3-L- triiodothyronine in myscdcma. Lancet l:ll 44l(l45, 1952.

This procedure is sensitive enough to allow reliable measurements of serum T not only in hyperthyroid patients but also in euthyroid and hypothyroid subjects. While this may be attributed in part to the use of a T -binding antiserum which usually allows detection of 0.03 ng of T in comparison to 0.1 ng T detected by the antiserum used previously known, the major improvement in sensitivity is a result of the use of nonthyroid hormone blocking agent e.g. ANS. Also. ANS has some advantages over compounds, such Dilantin and tetrachlorthyronine including better solubility at pH of RIA. lower cost and ready availability.

A mean serum T concentration of l 13 ng per ml in euthyroid subjects obtained by the use of the present method is comparable to 120. l 10 and ng per 100 ml observed by some other investigators. using another RIA of T However, it is believed that the difference in the mean normal serum T of 138 ng per 100 ml reported by these investigators, (Mitsuma et al.,: Radioimmunoassay of triiodothyronine in unextracted human serum. J. Clin. Endocr. 33:364-367, l97l and l 13 ng per 100 ml obtained by this procedure is indicative of random sample variation.

It is concluded that measurement of serum "l" by RIA affords an adequate separation of hyperthyroid and hypothyroid patients from normal subjects.

EXAMPLES l5-26 EXAMPLE COMPOUND 15 ANS lb ANNDS l7 TNBS l8 aNapthalcne Sullonic acid I) 5.5 diphenyl-Z thiohydantoin 2t! Doxepin HCl 2 l Diazepam Z1 ProchlorperaYine 23 Thimerosal 24 Sodium Salicylatc 25 Halolenatc (MK-] 2r Chlorpromazine HCl Proprietary compound supplied by Merck Sharp & Dohmc While various modifications of the invention have been herein described, various modifications of this invention will become apparent to those skilled in the art, and the scope of the invention is to be determined by the claims which follow.

l claim:

l. A method of measurement of the concentration of a particular thyroid hormone selected from the group consisting of thyroxine (T and triodo-L-thyronine (T in unextracted human serum. which comprises:

A: adding, to a measured quantity of unextracted human serum,

a. a blocking agent in an amount sufi'icient to displace essentially all of said particular thyroid hormone to be measured from thyroxine-binding globulin (TBG),

b. buffering ions to buffer said serum to a pH of between about 68 to about 9.6,

c. radioactive thyroid hormone, of the type to be measured. in an amount which will give a measurable counting rate of either antibody bound or free radioactivity after reaction equilibrium has been reached as set forth in Step B below; and

d. an antibody in sufficient quantity to bind a significant quantity of said radioactive thyroid hormone in the absence of any of the particular nonradioactive thyroid hormone to be measured,

8: allowing reaction of both particular thyroid hormone, to be measured, and said radioactive thyroid hormone, with said antibody to proceed substantially to equilibrium to thereby produce antibody bound radioactive thyroid hormone;

C: separating said antibody bound radioactive thyroid hormone, to be measured from said free radioactive thyroid hormone;

D: measuring the quantity of radioactive thyroid hormone selected from antibody bound radioactive thyroid hormone and free radioactive thyroid hormone;

E: preparing a standard curve with known amounts of the particular thyroid hormone to be measured, and

F: correlating the quantity of radioactive thyroid hormone measured with a known amount of said particular thyroid hormone read from said standard curve.

2. The method of claim 1 wherein said thyroid hormone to be measured is thyroxine, said blocking agent is 8-anilinol-napthalene disulfonic acid, said blocking agent being added to said human serum in quantities of at least about 4 ug/ul serum.

3. The method of claim I wherein said buffering ions are barbital ions, and said pH of said serum is about 8.6.

4. The method of claim I wherein said radioactive thyroid hormone is initially present in the order of [(1,000 counts per minute.

5. The method of claim I wherein said antibody bound radioactive thyroid hormone after reaction, gives a counting rate of from about 2()006000 counts per minute.

6. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with a second antibody.

7. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with gamma globulin.

8. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with charcoal suspension 9. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with polyethylene glycol.

10. The method of claim I wherein said thyroid hormone to be measured is T said blocking agent is 2.7 naphthalene disulfonic acid, said blocking agent being added to said human serum in quantities of at least about 2 ug/ul serum.

11. The method of claim 1 wherein said thyroid hormone to be measured is T;,, said blocking agent is 2,7 naphthalene disulfonic acid and said blocking agent is added to said human serum in quantities of at least about 1 ug/ul serum.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. ,09

DATED October 7, 1975 INVENTOMS) i CHOPRA, INDER J.

it is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown beiow;

Column 13, line 26, "disulfonic" should read:

sulfonic;

Column 14, line 26, prior to "2,7" insert;

3(4 anilino-l-napthylazo) line 31, prior to "2 ,7" insert:

3-(4 anilino-l-napthylazo) fligncd and Scaled this thirtieth Day of number 1975 [SEAL] Arrest:

RUTH C. MASON C. IAISIIALL DANN Arresting Officer (ommmiuncr a! hklls and Trademark: 

1. A METHOD OF MEASUREMENT OF THE CONCENTRATION OF A PARTICULAR THYROID HORMONE SELECTED FROM THE GROUP CONSISTING OF THYROXINE (T4) AND TRIODO-L-THYRONINE (T3) IN UNEXTRACTED HUMAN SERUM, WHICH COMPRISES: A: ADDING, TO A MEASURED QUANTITY OF UNEXTRACTED HUMAN SERUM, A. A BLOCKING AGENT IN AN AMOUNT SUFFICIENT TO DISPLACE ESSENTIALLY ALL OF SAID PARTICULAR THYROID HORMONE TO BE MEASURED FROM THYROXINE-BINDING GLOBULIN (TBG), B. BUFFERING IONS TO BUFFER SAID SERUM TO A PH OF BETWEEN ABOUT 6,8 TO ABOUT 9.6, C RADIOACTIVE THYROID HORMONE, OF THE TYPE TO BE MEASURED, IN AN AMOUNT WHICH WILL GIVE A MEASURABLE COUNTING RATE OF EITHER ANTIBODY BOUND OR FREE RADIOACTIVITY AFTER REACTION EQUILIBRIUM HAS BEEN REACHED AS SET FORTH IN STEP B BELOW, AND D. AN ANTIBODY IN SUFFICIENT QUANTITY TO BIND A SIGNIFICANT QUANTITY OF SAID RADIOACTIVE THYROID HORMONE IN THE ABSENCE OF ANY OF THE PARTICULAR NON-RADIOACTIVE THYROID HORMONE TO BE MEASURED, B: ALLOWING REACTION OF BOTH PARTICULAR THYROID HORMONE, TO BE MEASURED, AND SAID RADIOACTIVE THYROID HORMONE, WITH SAID ANTIBODY TO PROCEED SUBSTANTIALLY TO EQUILIBRIUM TO THEREBY PRODUCE ANTIBODY BOUND RADIOACTIVE THYROID HORMONE: C: SEPARATING SAID ANTIBODY BOUND RADIOACTIVE THYROID MONE, TO BE MEASURED FROM SAID FREE RADIOACTIVE THYROID HORMONE, D: MEASURING THE QUANTITY OF RADIOACTIVE THYROID HORMONE SELECTED FROM ANTIBODY BOUND RADIOACTIVE THYROID HORMONE AND FREE RADIOACTIVE THYROID HORMONE, E: PREPARING A STANDARD CURVE WITH KNOWN AMOUNTS OF THE PARTICULAR THYROID TO BE MEASURED, AND F: CORRELATING THE QUANTITY OF RADIOACTIVE THYROID HORMONE MEASURED WITH A KNOWN AMOUNT OF SAID PARTICULAR THYROID HORMONE READ FROM SAID STANDARD CURVE.
 2. The method of claim 1 wherein said thyroid hormone to be measured is thyroxine, said blocking agent is 8-anilino-1-napthalene disulfonic acid, said blocking agent being added to said human serum in quantities of at least about 4 ug/ul serum.
 3. The method of claim 1 wherein said buffering ions are barbital ions, and said pH of said serum is about 8.6.
 4. The method of claim 1 wherein said radioactive thyroid hormone is initially present in the order of 10,000 counts per minute.
 5. The method of claim 1 wherein said antibody bound radioactive thyroid hormone after reaction, gives a counting rate of from about 2000-6000 counts per minute.
 6. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with a second antibody.
 7. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with gamma globulin.
 8. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with charcoal suspension.
 9. The method of claim 1 wherein said antibody bound radioactive thyroid hormone, to be measured, is separated from said free radioactive thyroid hormone by precipation of said antibody bound radioactive thyroid hormone with polyethylene glycol.
 10. The method of claim 1 wherein said thyroid hormone to be measured is T4, said blocking agent is 2,7 naphthalene disulfonic acid, said blocking agent being added to said human serum in quantities of at least about 2 ug/ul serum.
 11. The method of claim 1 wherein said thyroid hormone to be measured is T3, said blocking agent is 2,7 naphthalene disulfonic acid and said blocking agent is added to said human serum in quantities of at least about 1 ug/ul serum. 